Changes of calcium ion concentration in keratinocytes are involved in regulation of skin barrier homeostasis and keratinocyte differentiation. Moreover, intracellular calcium dynamics might play a role in skin sensation.… We previously showed that exposure of cultured keratinocytes to mechanical stresses induces intracellular calcium elevation and intercellular calcium propagation, however, calcium dynamics in human epidermis is still poorly understood. In this study, we demonstrated a novel method for real-time measurement of calcium dynamics in response to point stimulation of human epidermis. Calcium propagation in cross-sectional samples of living human epidermis ex vivo was measured by two-photon microscopy after cells in stratum granulosum were stimulated by emission laser of the microscopy. Cells in stratum basale showed the greatest elevation of intracellular calcium. Calcium propagation from stratum granulosum to stratum basale was inhibited in the presence of apyrase, which degrades ATP (adenosine triphosphate), or gap junction blockers. Calcium propagation of cultured keratinocytes was also observed. In cultured keratinocytes, calcium propagated in a concentric wave-like manner from the stimulation site, and propagation was strongly suppressed by apyrase. These results suggested that ATP and/or gap junctions might play an important role in calcium propagation induced by point laser stimulation of the uppermost layer of epidermis. Our method should be broadly useful to study calcium dynamics, epidermal physiological mechanisms, and mechanisms of skin sensation at the single-cell level.